Posttranslational modification by ubiquitin and ubiquitin-like protein is essential for protein quality control. Defects in ubiquitin-mediated proteolysis are pathogenic to various cardiac diseases including desmin-related cardiomyopathy (DRC) and myocardial ischemia reperfusion (I/R) injury. However, little is known about the importance of ubiquitin-like proteins such as NEDD8 in cardiac myocytes (CM) physiology and pathophysiology. Preliminary data in this application reveal a novel and underappreciated role of NEDD8 in the CM and form the basis of our long-term goal: to understand how NEDD8 signaling can be modulated to regulate CM function and ultimately cardiac disease. Conjugation or deconjugation of NEDD8 to target proteins (neddylation or deneddylation) is dynamically regulated by a family of enzymes. Neddylation regulates a variety of essential cellular processes including proteolysis. Our preliminary data reveal a striking increase of neddylated proteins in the hearts from animal models of cardiac diseases, as well as in human failing hearts with ischemic or dilated cardiomyopathy. The buildup of neddylated proteins is functionally significant, as the mice deficient of a NEDD8 deconjuation enzyme develop severe cardiomyopathy through impairing proteolysis. Attenuation of neddylation by multiple means protects CMs from cell injury. These findings collectively indicate that the excessive neddylated proteins impairs proteolysis and are toxic to CMs, and that strategies to prevent/normalize neddylated proteins promote CM survival and function. NUB1L is a negative regulator of neddylation. NUB1L expression reduces neddylated proteins, enhances proteasomal function, and prevents stress-induced CM cell death. Therefore, the goal of the current project is to define the role of NUB1L in regulation of neddylation and protein degradation in the heart. We will test our central hypothesis that NUB1L reduces neddylated proteins and protects the heart against proteotoxic stress. Aim 1 will determine how NUB1L regulates neddylation and the impact of diminished neddylation on cardiac structure and function, using a novel transgenic mouse model. Aim 2 will test whether NUB1L expression enhances cardiac proteasomal function using a transgenic reporter, and determine whether suppression of neddylation regulates the removal of misfolded proteins in the heart and alters DRC progression. Aim 3 will determine the efficacy of suppression of neddylation, by both pharmacologic inhibitor and NUB1L expression, on myocardial I/R injury. Strategies to reduce neddylation may be effective therapeutic strategies to treat DRC and ischemic cardiomyopathy. The translational outcomes of this proposal include the preservation of muscle in myocardial infarction and prevention of heart failure, key missions of the NIH. The proposed studies are the first to target protein neddylation in models of cardiac diseases and will employ an innovative approach using genetically modified mice and pharmacologic intervention.